key: cord-0841597-2uqp7hu1
authors: Simons, Peter; Rinaldi, Derek A.; Bondu, Virginie; Kell, Alison M.; Bradfute, Steven; Lidke, Diane; Buranda, Tione
title: Integrin activation is an essential component of SARS-CoV-2 infection
date: 2021-07-21
journal: bioRxiv
DOI: 10.1101/2021.07.20.453118
sha: 69f9eced330d24cc465125d0818e2c7c2a27d5fa
doc_id: 841597
cord_uid: 2uqp7hu1

Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally: ‘inside-out’ signaling primes the ligand binding function of integrins via a talin dependent mechanism and ‘outside-in’ signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and Gα13 binding to the cytoplasmic tail of an integrin’s β subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.

To further investigate the role of integrins in SARS-CoV-2 R18 entry into Vero E6 cells, we used high binding affinity integrin antagonists: 1) BTT 3033, a selective antagonist (EC50 = 130 nM) of integrin α2β1 that binds to a site close to the a2I MIDAS domain and stabilizes the integrin bent conformation state (BCS), 46 2) ATN-161, a non-RGD peptide 47 derived from the synergy region of fibronectin, 48 known to exhibit specific antagonism for a5b1 and aIIbb3 and also recently shown to inhibit SARS-CoV-2 infectivity, 26 and 3) GLPG0187, a high-affinity, broad-spectrum (EC50 <10 nM) integrin receptor antagonist of RGD integrins a5b1, avb3, avb5, avb1, avb6. 49 We used a titrated, 5-fold excess of unlabeled SARS-CoV-2 relative to fluorescent SARS-CoV-2 R18 as a control for competitive inhibition of SARS-CoV-2 R18 binding.

Paired samples of cell suspensions in Mn 2+ -replete and Mn 2+ -free media were treated with the above integrin antagonists. Total viral binding was normalized to Mn 2+ -treated samples for each experimental condition. Mn 2+ treatment increased SARS-CoV-2 R18 occupancy of cells by ~20% compared to Mn 2+ -free conditions ( Fig. 3A-C) . The positive control for inhibition (5xCov-2 in data graphs) blocked 80% of SARS-CoV-2 R18 and was agnostic to Mn 2+ treatment. Reasoning that the residual signal of 5x Cov-2 treated samples was due to non-specific binding to the cell membrane, we subtracted the fluorescence of cells blocked with 5xCov-2 and then normalized the data to mock treated cells. We compared the relative efficacy of the inhibitors in Mn 2+ -replete and -free conditions of the normalized data ( Fig. 3D-F) . The fraction of Mn 2+ -activated integrins (20%) were refractory to BTT 3033 treatment (Fig.  3D) . BTT 3033 selectively binds to the BCS integrin structure 46 and does not bind to Mn 2+ activated integrins. In contrast, ATN-161 and GLPG0187 were agnostic to Mn 2+ treated cells, as the same baseline was achieved for either condition (Fig. 3D-F) . The inhibition is subtly better in the presence of Mn 2+ due to higher affinity for the RGD-targeting inhibitors. Overall, GLPG0187 was a better competitive inhibitor of SARS-CoV-2 R18 compared to ATN-161. The mechanistic specificity of integrin inhibition by these antagonists (BTT 3033 vs. GLPG0187) in regards to SARS-CoV-2 uptake strongly supports the ideas that: 1) integrin RGD engagement is an essential co-factor for cell entry and 2) integrin extension is required for cell entry based on BTT 3033's mechanism of action.

We then used live-cell confocal microscopy to visualize Vero E6 cell entry and trafficking of SARS-CoV-2 R18 in DMSO (mock)-and BTT 3033 -treated cells (Fig. 4A, B) . Most of the cells treated with GLPG0187 de-adhered from the plate and were thus not suitable for imaging. The loss of cells with GLPG0187 was likely due to the loss of integrin-mediated adhesion by the broad-spectrum inhibitor. Cells were imaged at 3-min intervals for 21 min after addition of ~10 7 SARS-CoV-2 R18 particles. In DMSO treated cells (Mock in Fig. 4) , SARS-CoV-2 R18 particles were visible at cell membranes within 3 minutes subsequently developed punctate features at the cell periphery and trafficked to the perinuclear space. The rate of cell entry (time to perinuclear space ~ 10 min) was comparable to infectious virions. 50 For the BTT 3033-treated cells, early peripheral membrane localization of SARS-CoV-2 R18 showed significant diminution of discernable puncta and did not undergo retrograde traffic towards the perinuclear region within the timeframe of the experiment. The relative amount of virus binding to the surface was also reduced with BTT 3033 treatment (Fig. 4A, B) , consistent with reduced binding observed by flow cytometry measurements (Fig. 2) .

Canonical, physiologic integrin activation is started by sequential waves of inside-out signaling initiated by talin binding to the b-subunit cytoplasmic tail (b-CT), which causes integrin extension (ECS in Fig.  1 ). 51 ECS integrin binding to immobilized ligand facilitates outside-in signaling as a force applied at the adaptor protein by the actin cytoskeleton is resisted at the ligand-binding site. 20, 21 Transmission of the tensile force through the integrin to the adaptor protein stabilizes high-affinity integrin binding (in the EOS) to the ECM. This induces the G protein subunit Ga13 to transiently replace talin 43 at the b-CT site, leading to cell spreading, retraction, migration, and internalization of the receptor 40, 52-54 (Fig. 5A) . The sequential mechanism of inside-out and outside-in was confirmed in part by the use of two myristoylated peptides; mP6 (Myr-FEEERA-OH), derived from the Ga 13 -binding ExE motif of integrin b-CT, and mP13 (Myr-KFEEERARAKWDT-OH) mimicking the b-CT's talin binding domain. 42 mP6 is known to suppress the early phase of outside-in signaling and mP13, which binds both talin and Ga13 blocks all phases of integrin signaling. 42 To investigate the relationship between the integrin signaling events and SARS-CoV-2 engagement and cell entry, we treated cells with mP6 peptide, which inhibited cell entry of SARS-CoV-2 R18 in flow cytometry and microscopy experiments (Fig. 5B-D) . Similarly, mP13 inhibited cell entry in flow cytometry experiments (data not shown). The results for mP6 treated cells (Fig. 5B-D) suggest that SARS-CoV-2 engagement initiates a Ga13-mediated outside-in integrin activation inhibited by mP6, as previously demonstrated for the Sin Nombre virus. 23

Because mP6 and mP13 are membrane-permeable peptides, they were suitable for infectivity experiments while obviating the need to expose cells to DMSO for extended periods. We therefore tested the efficacy of mP6 and mP13 at inhibiting cell entry and productive infection in Vero E6 cells with a 0.01 multiplicity of infection (MOI) of SARS-CoV-2. For the productive infection assay, infected cells were plated at confluency (500,000 cells/well in a 12 well plate) to minimize cell growth for 48 hrs postinfection. We used RT-qPCR to measure viral RNA in the suspended cells or intact cell monolayers at 48hrs post-infection, respectively. At 48 hrs post-infection, inhibition of productive infection by mP13 was significant relative to mock-treated cells, whereas the effect of mP6 was insignificant (Fig. 5E) . These results suggest the binding of Ga13 to b3 integrin induced by SARS-CoV-2 in resting cells (Fig. 5B) is a dispensable mechanism of integrin activation under the prevailing conditions of cells perturbed by viral replication. 23 Stated differently, the primary infection was conducted with quiescent cells, in which Ga13dependent integrin activation was initiated by SARS-CoV-2 engagement. We suggest that in postinfection cells, the Ga13-mediated signaling is expendable, in contrast to talin-mediated signaling (see discussion). 23 Thus, the efficacy of mP13 as an inhibitor of productive infection suggests that integrins play an enduring role in the lifecycle of SARS-CoV-2. 17, 24, 28 

This study provides mechanistic evidence for the functionality of extracellular ligand-binding domains of integrin b1 and cytoplasmic tails of integrins in general, 24, 28 which offer possible molecular links between ACE2 and integrins. We show that Mn 2+ , which induces integrin extension and high affinity ligand binding, enhances the cell entry of SARS-CoV-2 R18 . This is consistent with the notion that integrin affinity and/or extension is an essential factor for cell entry. In support of integrin-dependent endocytosis as a pathway of SARS-CoV-2 R18 internalization, we used broad-spectrum RGD antagonists such as GLPG0187, which inhibited cell entry regardless of integrin activation status. Our study also suggested integrin specificity. BTT 3033, an aI allosteric antagonist that binds to the bent closed conformation of integrin b1 and stabilizes it, supports the possibility of integrin-dependent endocytosis of SARS-CoV-2 R18 upon receptor binding. In a different framework, our data also show that SARS-CoV-2 R18 can bind to low affinity and presumptively bent-conformation integrins, 22 however, in BTT 3033 treated cells, cell entry by SARS-CoV-2 R18 is inhibited because integrin activation post-SARS-CoV-2 R18 engagement is prevented. Thus, our data contextualize integrin extension as the "sine qua non of integrin cell adhesion function," 22 which in turn is an essential condition for integrin-mediated cell entry by SARS-CoV-2.

The binding of adaptor proteins such as talin and Ga13 to the integrin b-subunit cytoplasmic tail are essential elements of inside-out and outside-in physiologic integrin signaling. 42 mP6 and mP13 both blocked initial cell entry of SARS-CoV2 18 . However, only mP13 inhibited productive infection. In an earlier study of Sin Nombre virus infectivity, we showed that artificially-induced discharge of intracellular Ca 2+ stores elicited integrin inside-out signaling function, which dispensed with the need for Ga13 mediated signaling. 23 Under our experimental conditions lasting 48 hours, we estimate that successive replication and release of progeny virions occurred every ≥8 hours. 50, 55 We suggest that the overall process of productive infection, is characterized by viral perturbation of Ca 2+ homeostasis within the infected cells. 56 Thus one might expect persistent integrin priming 57, 58 which may render the Ga13 pathway to be redundant.

Mészáros et al. 24 have used bioinformatics to predict the existence of short amino acid sequences (~3-10 residues): short linear motifs (SLiMs), in the cytoplasmic tails of ACE2 and integrins that mediate endocytosis and autophagy. Some of their theoretical predictions have been validated by experimental studies. First, Kliche et al. 28 confirmed the existence of SLiMs. They extended their findings to establish a potential connection between ACE2 and integrin b3 cytoplasmic tail interactions with scaffolding and adaptor proteins linked to endocytosis and autophagy. Second, SLiM sequences known to bind and activate the transmembrane glycoprotein neuropilin 1 (NRP1) were identified as potential mediators of SARS-CoV-2 endocytosis. 24 Interestingly, NRP1, which is abundantly expressed in the olfactory epithelium is now declared as an effector for SARS-CoV-2 infection. 34, 35 NRP1 localizes at adhesion sites and promotes fibronectin-bound, activated a5b1 integrin endocytosis and directs the cargo to the perinuclear cytoplasm. [29] [30] [31] [32] [33] [34] Studies have shown that the endocytosis of active and inactive integrins to EEA1-containing early endosomes follows distinct mechanisms involving different adaptor proteins. The inactive integrin is promptly recycled back to the plasma membrane via an ARF6-and EEA1-positive compartment in a Rab4 -dependent manner. 31 We observed that in BTT 3033-treated cells replete with inactive β1 integrins, SARS-CoV-2 R18 remained membrane-bound, whereas untreated cells displayed internalization and perinuclear localization of SARS-CoV-2 R18 . This is consistent with the known trafficking of active integrins, including those directed by NRP1, to the perinuclear space. 29, 30, 32 Although several integrin types 24-28 are believed to be co-receptors of SARS-CoV-2 infectivity, our study suggests inhibitor specificity for integrin b1. This is consistent with known factors: 1) correlated increased expressions of b1 14 and ACE2 in relevant tissues, 15, 16 2) cytoplasmic tail in cis interactions between ACE2 and integrin b1, 13 and 3) synergy between ACE2 and integrin b1 signaling that promotes RGD mediated cell adhesion. 17 To optimize integrin engagement, our cell-binding assays and primary infection assays were carried out in suspension such that ACE2 and integrins were not segregated by cell polarization. 59, 60 However, our microcopy studies on adherent cells were in agreement with the flow cytometry results. Thus, our study represents an initial step forward in establishing a mechanistic role for SARS-CoV-2-mediated integrin activation required for cell entry and productive infection.

USA-WA1/2020 SARS-CoV-2 strain was obtained from BEI Resources (NIAID, NIH). Integrin inhibitors, BTT3033, a selective inhibitor of a2b1, ATN-161 an integrin a5b1 antagonist, 26 and GLPG0187 a broad-spectrum integrin inhibitor, were purchased as powders from Tocris Bioscience. The EEA1 rabbit monoclonal antibody (clone C45B10) was from Cell Signaling Technologies (CAT# 3288S). Alexa fluor 647 conjugated F(ab')2 fragment goat anti-rabbit IgG was from Invitrogen (CAT# A21246). In addition, myristoylated peptides; mP6 (Myr-FEEERA-OH) and mP13 (Myr-KFEEERARAKWDT-OH) were custom synthesized at Vivitide.

African green monkey kidney cells (Vero E6, ATCC) were maintained in DMEM media from Sigma CAT# D5796. All media contained 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine and were kept at 37 °C in a CO2 water-jacketed incubator of 5% CO2 and 95% air (Forma Scientific, Marietta, OH, USA). UV inactivation and fluorescent labeling of the envelope membrane of SARS-CoV-2 with octadecyl rhodamine (R18).

USA-WA1/2020 SARS-CoV-2 strain (from BEI Resources, NIAID, NIH) was cultured in Vero E6 cells in a biosafety level 3 (BSL-3) containment under a protocol approved by the University of New Mexico's Institutional Biosafety Committee or IBC (Public Health Service registration number C20041018-0267). Live SARS-CoV-2 were harvested at peak titers of 10 7 plaque-forming units/mL (PFU/ml). Next, SARS-CoV-2 was U.V. inactivated using 254 nm (≈ 5 mW/cm 2 ) U.V. irradiation of a TS-254R Spectroline UV Transilluminator (Spectronics Corp., Westbury, NY) following a similar protocol for inactivating pathogenic orthohantaviruses. 45, 61 Briefly, Vero E6 cells were inoculated with SARS-CoV-2 and maintained at 37°C for 2-4 days. At 70-75% cell death (due to viral cytopathic effect), the supernatant was harvested and subjected to light centrifugation (1000 rpm, 10 min) to remove cellular debris. For U.V. inactivation, supernatants were added to a 12 well plate at 500 µl aliquot/well. Then U.V. irradiated at 3.8 cm above the sample for 0, 10,15, 20, 25, 30, 60, and 90 seconds and then tested for viability by a three-day plaque assay as described elsewhere. 62, 63 The titration of U.V. irradiation times was used to establish a minimal U.V. dose for complete inactivation. After U.V. treatment, the 500 µl fractions were pooled into 15 mL tubes stored in a -80°C freezer pending the results of a plaque assay. Under our experimental conditions, we established that a minimum U.V. irradiation interval of 25 seconds was required for the complete inactivation of SARS-CoV-2. A 90 sec UV dose was approved by the IBC for removal of inactivated SARS-CoV-2 out of the BSL-3 lab after it was established that the virus particles were capable of specific binding to Vero E6 cells.

Crude UV-inactivated SARS-CoV-2 samples were purified by floating 10 ml of Vero E6 SARS-CoV-2 supernatant on a density gradient comprising 2 ml volumes of 1.2 g/ml and 1.0 g/ml CsCl in PBS media in 14 × 89-mm Beckman polyallomer tubes. The samples were centrifuged for 1.5 hours at 4 °C using a Beckman SW41Ti rotor at 30,000 pm. Individual fractions were collected, and a refractometer was used to identify the fraction that contained SNV by its putative density of 1.0 g/ml. The purified SARS-CoV-2 samples were stored in 1.0 ml aliquots at -80 °C. SARS-CoV-2 particles were fluorescently labeled and calibrated according to the same protocol used for the Sin Nombre virus (SNV). 45 SARS-CoV-2 R18 particles were stored at -80°C in 20 µl (2x10 8 particles/µl) aliquots.

For flow cytometry assays, cells were cultured in T25 or T75 flasks to 80% confluence. Cells were then treated with 0.25% trypsin and then transferred to minimum essential medium (MEM) media. Test suspension cell samples were transferred to microfuge tubes in 40 µl-aliquots (1,000 cells/µl). SARS-CoV-2 R18 was added to tubes at 5,000 SARS-CoV-2 R18 /cell and incubated using a shaker at 500 rpm for 20 min at 37°C. For blocking assays, cells were incubated with 5x unlabeled SARS-CoV-2 or 10 µM integrin inhibitors for 20 min before the addition of SARS-CoV-2 R18 . Samples were centrifuged at 3,000 rpm; the pellet was resuspended in HHB buffer (30 mM HEPES, 110 mM NaCl, 10 mM KCl, 1 mM MgCl2•6H2O, and 10 mM glucose, pH 7.4) buffer and read on an Accuri flow cytometer. For kinetic assays, Vero E6 suspension cells in 40 µl volumes (1,000 cells/µl) were placed in ±Mn 2+ media in duplicate microfuge tubes at 37°C. Sars-CoV-2 R18 was then added (5,000 virions/cell) to the tubes and incubated for 1, 3, 5, 7, 9 min. At each time point, the tubes were quenched in an ice bath, then samples were centrifuged and resuspended in 95 µl HHB buffer and analyzed on a flow cytometer Live Cell Confocal Microscopy.

Imaging was performed using a Leica TCS SP8 Laser Scanning Confocal Microscope with a 63× water objective and a Bioptechs objective heater to maintain cells at physiological temperature (~36-37 °C). Vero E6 cells were plated in eight-well Lab-Tek (Nunc) chambers at a density of 30,000 cells per well 24 hours before imaging. Cells were imaged in Tyrode's buffer (135 mM NaCl, 10 mM KCl, 0.4 mM MgCl2, 1 mM CaCl2 20 mM glucose, 0.1% BSA, 10 mM HEPES, pH 7.2). For integrin inhibition, cells were treated with 10 μM BTT3033-α2β1 or 50 μM MP6 in Tyrode's buffer for 30 minutes before imaging. ~1x10 9 SARS-CoV-2 R18 particles were added per well and z-stacks (300 nm thickness) were acquired every 3 minutes for 21 minutes to visualize viral cell entry. R18 was excited using 561 nm light, isolated from the white light source. R18 emission and differential interference contrast (DIC) transmitted light were captured with Leica Hybrid detectors (HyD) in a spectral window of 571-636 nm (for R18 emission). Analysis of the accumulation of SARS-CoV-2 R18 particles in Vero E6 cells was completed using Matlab. Briefly, regions of interest (ROI) were created around the cell membrane and the mean SARS-CoV-2 R18 intensity was measured within the cell mask at each time point.

Vero E6 cells were plated on 18-mm coverslips overnight in a 6 well plate at a density of 100,000 cells/well. Cells were exposed to ~1x10 9 SARS-CoV-2 R18 particles/well for 15 min at 37 °C, in the presence or absence of 10 μM BTT 3033. Cells were then washed in phosphate-buffered saline (PBS) and fixed using 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature. Cells were extensively washed with 10 mM Tris (pH 7.4) and PBS and permeabilized with 0.1% Triton. Cells were labeled with anti-EEA1 primary antibody and anti-rabbit Alexa Fluor 647 secondary. Nuclei were stained with Hoechst 33258. Cells were mounted on microscope slides using Prolong Diamond Antifade Mountant (Invitrogen, CAT#P33970). Samples were imaged using a TCS SP8 Laser Scanning Confocal Microscope with a 63× oil objective.

Vero E6 cells grown at 80% confluency were trypsinized and divided into microfuge tubes aliquots of 1.5x10 6 cells in 750 µl media containing 250µM mP6, 250µM mP13, 10µM BTT 3033, DMSO, and media only. Samples were shaken at 500 rpm for 30 min at 37°C. After transfer to a BSL-3 laboratory, 0.01 MOI of SARS CoV-2 (lot #P3: 1.2x10 7 pfu) and then incubated for 60 min while shaking. Tubes were spun down (1000rpm for 3 min), resuspended in fresh media, and spun down again. The cells were then resuspended in 300 µl of media and transferred to a 12 well plate in 100 µl aliquots (500,000 virions/well) for triplicate measurements. An additional 400µl were added to each well for a final volume of 500 µl. The plate was transferred to an incubator for 48 hrs to allow the virus to replicate. The cells were then washed with 1xPBS before RNA was extracted with TRIzol TM (Thermofisher, #15596026) according to the manufacturer's protocol: (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/trizol_reagent.pdf). 

Duplicate plaque assays of supernatants of Sars-CoV-2 exposed to increasing doses of 254 nm radiation and then tested for viability. The live virus completely lysed the cells at 1:100 dilution relative to U.V. exposed virions. B. Graph shows UV dose response, leading to a significant decrease in plaque forming units at different doses. For our experiments, 90 second (450 mW•sec/cm 2 ) U.V. dose was used to inactivate the virus prior to removal from the BSL-3 laboratory. C. Binding kinetics of SARS-C0V-2 R18 to suspension Vero E6 cells at 37°C in Ca 2+ and Ca 2+ /Mn 2+ replete cells. D. Equilibrium binding of SARS-CoV-2R18 after 30 min incubation at 37°C. E. Confocal microscopy imaging of cells after incubation with SARS-CoV-2 R18 (magenta) for 15 min, then fixed and labeled for early endosome marker, early endosome antigen 1 or EEA1 (green) an effector protein for Rab5, and nuclei (Hoechst 33258, blue). SARS-CoV-2 R18 vesicles are trafficked to the perinuclear region and a subset are co-localized with EEA1. Images are maximum projections and have been brightness and contrast enhanced. Model of outsideinside-out signaling for integrin-mediated cell entry. Hypothetical SARS-CoV-2 binding to integrin b1 initiates Ga13 binding to the b1 cytoplasmic tail, which stimulates outside-in signaling in the absence of a known receptor-stimulated GPCR mediated inside-out signaling. mP6 is a specific inhibitor of Ga13 binding to the b1 cytoplasmic tail. B. Relative fluorescence readings of suspension Vero E6 cells after 30 min incubation with SARS-CoV-2 R18 in vehicle and 100 µM mP6 treated cells. C. Live cell imaging of SARS-CoV-2 R18 (magenta) binding and endocytosis shows cell membrane and perinuclear localization of SARS-CoV-2 R18 vesicles while virus is seen to remain at the plasma membrane in cells treated with 50 µM mP6. LUT ranges shown in the bottom left corner of 6-min timepoint images. Scale bars, 10 µm. D. Traces of absolute intensity values of virus binding over time. Two representative cells for each condition are plotted from data acquired on the same day to enable direct comparison of intensity values. For comparison, mock-treated cell data are the same as in Figure 4 . Data were fit to a non-linear regression function with arbitrary constants for appearance purposes. E. Inhibition of SARS-CoV-2 productive infection. Suspension Vero E6 cells were preincubated with 250 µM mP6 and 250 µM mP13 for 30 min and followed by infection with 0.01 MOI SARS-CoV-2 for an additional 60 min incubation. Cells were washed twice and transferred to a 12 well plate for 48 hours and assayed for viral RNA by RT-qPCR.

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Cell integrins: commonly used receptors for diverse viral pathogens

Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues

The protein expression profile of ACE2 in human tissues

Mechanisms of SARS-CoV-2 Transmission and Pathogenesis

Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein

Interaction of ACE2 and integrin beta1 in failing human heart

Temporal response and localization of integrins beta1 and beta3 in the heart after myocardial infarction: regulation by cytokines

Deficiency of beta1 integrins results in increased myocardial dysfunction after myocardial infarction

Trilogy of ACE2: a peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters

Angiotensin converting enzyme (ACE) and ACE2 bind integrins and ACE2 regulates integrin signalling

Integrin structures and conformational signaling. Current opinion in cell biology

Structural basis of integrin regulation and signaling

Coordinated integrin activation by actin-dependent force during T-cell migration

Regulation of integrin affinity on cell surfaces

Measuring Integrin Conformational Change on the Cell Surface with Super-Resolution Microscopy

Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications

The Spike Glycoprotein of SARS-CoV-2 Binds to beta1 Integrins Expressed on the Surface of Lung Epithelial Cells

The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection

SARS-CoV-2 uses major endothelial integrin alphavbeta3 to cause vascular dysregulation in-vitro during COVID-19

Cytoplasmic short linear motifs in ACE2 and integrin beta3 link SARS-CoV-2 host cell receptors to mediators of endocytosis and autophagy

Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells

Conformationally active integrin endocytosis and traffic: why, where, when and how?

Distinct recycling of active and inactive beta1 integrins

Integrin traffic -the update

The ESCRT machinery mediates polarization of fibroblasts through regulation of myosin light chain

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

Neuropilin-1 is a host factor for SARS-CoV-2 infection

Reconstruction of integrin activation

Integrin activation

Regulation of integrin activation

Complete integrin headpiece opening in eight steps

Inside-out, outside-in, and inside-outside-in: G protein signaling in integrin-mediated cell adhesion, spreading, and retraction

Regulation of integrin activity and signalling

A directional switch of integrin signalling and a new anti-thrombotic strategy

G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin "outsidein" signaling

Real-time partitioning of octadecyl rhodamine B into bead-supported lipid bilayer membranes revealing quantitative differences in saturable binding sites in DOPC and 1:1:1 DOPC/SM/cholesterol membranes

Recognition of decay accelerating factor and alpha(v)beta(3) by inactivated hantaviruses: Toward the development of high-throughput screening flow cytometry assays

Novel alpha2beta1 integrin inhibitors reveal that integrin binding to collagen under shear stress conditions does not require receptor preactivation

Pharmacology of the novel antiangiogenic peptide ATN-161 (Ac-PHSCN-NH2): observation of a U-shaped dose-response curve in several preclinical models of angiogenesis and tumor growth

The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes

A dose escalating phase I study of GLPG0187, a broad spectrum integrin receptor antagonist, in adult patients with progressive high-grade glioma and other advanced solid malignancies

SARS-CoV-2 (COVID-19) by the numbers

Ligand binding to integrins

Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling

Regulation of outside-in signaling and affinity by the beta2 I domain of integrin alphaLbeta2

Requirement of alpha and beta subunit transmembrane helix separation for integrin outside-in signaling

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

Viral calciomics: interplays between Ca2+ and virus

Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways

Signaling during platelet adhesion and activation. Arteriosclerosis, thrombosis, and vascular biology

Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus

Equilibrium and Kinetics of Sin Nombre Hantavirus Binding at DAF/CD55

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Evaluation of inactivation methods for severe acute respiratory syndrome coronavirus in noncellular blood products

Structures and distributions of SARS-CoV-2 spike proteins on intact virions

Supramolecular Architecture of the Coronavirus Particle

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

This project is supported by an award from the National Center for Advancing Translational Sciences, National Institutes of Health under grant number UL1TR001449, NIH R35GM-126934 (DSL), Fluorescence microscopy was performed in the University of New Mexico Comprehensive Cancer Center fluorescence microscopy shared resource (NIH P30CA118100).